Fig. 3

NOS2 deficiency leads to increased demyelination in the spinal cords of RSA59 infected mice at the acute-adaptive transition phase (day 9/10 p.i.) concurrent with increased expression of phagocytic markers. 5 µm thick transverse sections of RSA59 infected (20,000 PFUs) WT and NOS2-/- mice spinal cords were stained for the presence of inflammatory lesions by H & E (A), demyelination by LFB (B) and activated MG/Mφ by Iba1 (C). Black arrows in (A) indicate the regions with myelitis. Blue arrows in (B) indicate the demyelinating plaques. Black boxed areas highlighted by black arrow represent higher magnification of grey matter area, and brown boxed areas highlighted by brown arrow represent higher magnification of white matter below the corresponding Iba1 stained spinal cord transverse-sections in (C). Scale bar 100 µm. (D) and (E) are the quantification of percent demyelination and percent Iba1 stained area, respectively (n = 4 to 5 mice per group. 1 to 6 sections corresponding to different regions of the spinal cord were analysed and each dot in the graph represent one section from one spinal cord). Results were expressed as Mean ± SEM. *Asterisk represents statistical significance calculated between infected WT and infected NOS2-/- mice using unpaired student’s t test with Welch’s correction. p < 0.05 was considered as significant. *p < 0.05. mRNA was isolated from brain and spinal cord tissue of mock and infected (20,000 PFUs) RSA59 WT and NOS2-/- mice. Transcript levels of CD206, P2Y6, TREM2 and Arg1 were analyzed for brain (F) and spinal cord (G) by qRT- PCR. Results were expressed as Mean ± SEM. (n = 3 to 5. The mRNA expression was calculated by ΔCT method and average expression per biological sample was plotted.)) *Asterisk represents statistical significance using Two- ANOVA with Tukey’s multiple comparisons test. p < 0.05 was considered as significant. *p < 0.05 and **p < 0.01